Efficient CRISPR-mediated mutagenesis in primary immune cells using CrispRGold and a C57BL/6 Cas9 transgenic mouse line
Item Type: | Article |
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Title: | Efficient CRISPR-mediated mutagenesis in primary immune cells using CrispRGold and a C57BL/6 Cas9 transgenic mouse line |
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Creators Name: | Chu, V.T. and Graf, R. and Wirtz, T. and Weber, T. and Favret, J. and Li, X. and Petsch, K. and Tran, N.T. and Sieweke, M.H. and Berek, C. and Kühn, R. and Rajewsky, K. |
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Abstract: | Applying clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9)-mediated mutagenesis to primary mouse immune cells, we used high-fidelity single guide RNAs (sgRNAs) designed with an sgRNA design tool (CrispRGold) to target genes in primary B cells, T cells, and macrophages isolated from a Cas9 transgenic mouse line. Using this system, we achieved an average knockout efficiency of 80% in B cells. On this basis, we established a robust small-scale CRISPR-mediated screen in these cells and identified genes essential for B-cell activation and plasma cell differentiation. This screening system does not require deep sequencing and may serve as a precedent for the application of CRISPR/Cas9 to primary mouse cells. |
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Keywords: | CRISPR/Cas9, sgRNA Design, Knockout Efficiency, Primary Cells, B Cells, Animals, Mice |
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Source: | Proceedings of the National Academy of Sciences of the United States of America |
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ISSN: | 0027-8424 |
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Publisher: | National Academy of Sciences |
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Volume: | 113 |
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Number: | 44 |
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Page Range: | 12514-12519 |
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Date: | 1 November 2016 |
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Official Publication: | https://doi.org/10.1073/pnas.1613884113 |
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PubMed: | View item in PubMed |
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